Growing ''A. thaliana'' in Cut Pipet Tips

Tip preparation

Day 1 - tip cutting

1. Cut p200 (yellow) tips from the bag
   1. You want to cut just under the lip (about 1/4 of the way down), saving the top cylindrical part and discarding the true tip portion
   2. Holding scissor blade flush with the lip, cut allowing the depth of the blade to dictate how much under the cylindrical part to cut
2. Fill TipOne boxes 80mL water, and insert yellow "inserts" (from used filter tip boxes) and place cut tips into the holes - it should look like a regular box of p200 tips at first glance
3. Autoclave tip boxes in liquid cycle
4. Move boxes to -20 freezer for at least 24 hours (more will never hurt!!!)

Day 2

1. Preheat the little water bath to 40C
2. Melt 0.8% MS Agar in 2 min increments at 50% Power in the microwave of science. Leave in small water bath for at least 20min. Move to hood.
3. Bring 1 tip box (at a time) to the hood from the freezer
4. Using a p1000 set to 1 mL, add one drop of MS agar to the bottom "corner"/side of each of the frozen tips. The droplet sized volume and extreme coldness from the ice and freezer should prevent the agar from falling through.
5. Start with 1-2 drops for each tip in the box, allow to solidify (probably within the time it takes to do all 96). Repeat, adding a drop or two at a time to each tip until a plug forms at the bottom. Some agar will drip through and it's okay! Just be patient with each addition.
6. Once a strong plug forms, you should be able to top of the agar so it just barely mounds (not too much or the cover will squish it - I know it is obnoxious I'm sorry)
7. Place box in 4C fridge until ready to seed

Note: 1-2 cut, sterilized, filled full tip boxes needed for "A" treatments (1 plant per plant box - 130 plants/generation); At least 14 full tip boxes needed for "B" treatments (9 plants per plant box - 1170 plants/generation)

Sterilizing and Planting the Seeds

Day 3

Note: Perform this in sterile conditions.

1. Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 1mL of 70% ethanol. Move to hood.
2. Remove ethanol by pipetting, add 1mL 50% bleach (500uL bleach, 500uL sterile milliQ). Soak for 10 min
3. Remove all bleach residue by rinsing 5-7 times with sterile milliQ water.
   1. Add 1 mL of sterile distilled water to epitube and invert once; allow seeds to settle, pipet off water and continue 5 more times.

1. Top of tips with agar if necessary. LEVEL SHOULD NOT BE LOWER THAN RIM OF THE TIP!
2. Add 1 mL sterile milliQ water to the sterilized seeds.
3. Use the p200 pipette set to 100uL to suction 1 seed onto or into pipet. Deposit 1 seed onto each tiny agar surface

 :Note: If having difficulty picking up a single seed transfer seeds and water to small petri dish and move the seeds around to spread out. Additionally, if the seed is not coming out of the pipette or is stuck on the tip add additional water to the pipette and then place it in the soil. 

1. Once all of the pipet tips have a seed, gently close the lid and wrap entire box in tin foil, and place the boxes of tip plant babies at 4°C for 4 days. This cold treatment, also called stratification, will improve the rate and synchrony of germination.

Day 7

1. After 4 days, unwrap and discard foil and move boxes to the grow room under 16/8 day/night cycle. Allow 2-3 days to germinate.

Inoculating and Moving Tips to Soil

Day 9

1. Fill plant boxes with 45g potting soil. Autoclave.

Day 10

1. Create slurry combining 20g soil (either from nature trail for Gen0 or previous generation) and 300mL sterile milliQ water in a large media prep bottle. Shake to homogenize.
2. In the hood, pre-wet each potting soil box with 15mL sterile milliQ water --> allow to set for 5-10min
3. Once water is absorbed into soil, add 10mL slurry to the soil --> allow to settle 10 min

1. Move boxes of germinated plant tips to the hood along with a small beaker of tweezers/forceps soaking in 70% ethanol
2. Wipe excess ethanol from forceps with a paper towel
3. Pick up a successfully germinated tip plant with the forceps and push into the potting soil, ensuring the top of the pipette tip is flush or lower than (just not above) the soil line.
4. Move back to grow room. Allow to grow for 14 days before selection